Project Summary Crystallization, followed by subsequent structure determination, is a major step in understanding the structure-function relationship of macromolecules. Understanding macromolecule structure has become a key part in the development of new pharmaceuticals, and is a major area of NIH research. Crystallization however is also the rate limiting step, despite technological efforts to automate the set-up and crystallization data acquisition processes. Macromolecule crystallization conditions are arrived at by screening experiments, where the target material is typically subjected to hundreds or even thousands of different chemical cocktails. In most cases screening experiments fail as they do not result in a crystal. We propose that screening experiments contain useful information about the target proteins behavior in response to the tested solution conditions. No screen or group of screens can systematically cover the combinatorial chemical space for protein crystallization, and we hypothesize that in the absence of clear positive hits scored results can be analyzed to determine these factors. The analysis method developed is called the Associated Experimental Design (AED) approach. The analysis identified the most significant factors and a 96 condition screen based on those factors is prepared for each protein and set up. In the (ongoing) Phase I effort the AED software is being progressively evolved, adding functions for aiding in prioritizing the screen factors employed for likely success in crystallization. The software is written to not duplicate input conditions for a given protein in the output; i.e., all output conditions are new combinations of high probability factors as determined from the analysis. The software has been tested with 23 proteins to date. Of the 5 proteins that did not give crystals upon initial screening, 2 gave crystals from screens developed on the basis of the AED analysis. Of the 18 remaining proteins, 72% gave as many or more crystals in the single AED based screen than were obtained in the 4 x 96 condition screens. One of these 18 proteins was the RrP41-RrP42 archaeal exosome catalytic core complex. Based on the Phase I results the AED method shows considerable promise. A major advantage of this approach is that it fits into existing practice, making use of existing materials, methods, and data routinely generated in crystallization screening. The AED software can be used with any imaging system that gives a scored assessment of the results for each trial, including manual scoring by a user with a simple low power microscope. The Phase I results also showed that it can be used with a reduced, more granular, scoring scale. Success with this approach will increase the number of hits generated and greatly reduce the time and effort required for macromolecule crystallization. The proposed Phase II effort is to build upon the successful approach developed in Phase I and further develop the analytical methods employed.